Resistance of vaccinia virus to rifampicin conferred by a single nucleotide substitution near the predicted NH2 terminus of a gene encoding an Mr 62,000 polypeptide

Abstract

Marker transfer procedures were used to locate the site of mutation in the genome of a previously characterized (B. Moss, E. N. Rosenblum, and P. Grimley, 1971), Virology 45, 135-148) rifampicin-resistant (RifR) vaccinia virus isolate. Starting with a cosmid library prepared from the mutant genome, recombination with successively smaller DNA fragments was shown to transfer drug resistance to wild-type vaccinia virus. In this manner, the mutation was mapped within a 485-bp DNA segment in the central region of the genome at the extreme right end of the HindIII D fragment. Nucleotide sequencing indicated that this DNA segment differed from the homologous region of wild-type DNA by a single C/G----A/T substitution. Sequencing of the flanking 2195 bp revealed two tandem nonoverlapping open reading frames (ORFs) encoding putative polypeptides of Mr 16,908 and 61,840. The RifR mutation resulted in a predicted glutamine----lysine change only 27 amino acids from the NH2 terminus of the longer ORF. A predicted asparagine to aspartic acid substitution, found in another RifR vaccinia virus mutant by J. Tartaglia and E. Paoletti (Virology 147, 394-404, 1985), mapped near the carboxyl terminus of the same ORF. These data suggest a model in which head-to-tail interaction between Mr 61,840 polypeptides occurs and in which rifampicin blocks virus assembly by preventing this association.